Process for extraction of beta-glucan from cereals and products obtained therefrom

ABSTRACT

A process for obtaining β-glucan from cereal grain, such as barley and oats. A β-glucan product obtained by the process. Uses of the β-glucan product as a food ingredient and for treating various diseases or disorders. The process includes the steps of forming flour from the cereal grain, mixing the flour with water to form a slurry of a process for obtaining β-glucan from cereal grain including forming flour from the cereal grain, mixing the flour with water to form a slurry of an aqueous solution of β-glucan and a solid residue, separating the aqueous solution from the solid residue, and removing water from the aqueous solution by evaporation or ultrafiltration or combinations thereof to form a β-glucan containing gel or solid.

FIELD OF INVENTION

[0001] This invention relates to a novel process for the extraction ofβ-glucan from cereals, such as barley and oats. The invention alsorelates to β-glucan products obtained from the process. The inventionfurther relates to uses of those products as food ingredients andtherapeutic agents.

BACKGROUND

[0002] The term “β-glucan” refers to those polysaccharides whichcomprise D-glucopyranosyl units which are linked together by (1→3) or(1→4) β-linkages. β-Glucans occur naturally in many cereal grains suchas oats and barley. The molecular weight of β-glucan molecules occurringin cereals is typically 200 to 2000 kiloDaltons.

[0003] β-Glucan is desirable as a food additive, for example, to imparttexture (“mouth feel”) to foods or useful as edible films for foodcoatings. β-Glucan may also be used to add bulk to foods and has theadvantage of having a neutral flavour.

[0004] β-Glucan is also desirable as a therapeutic agent. There isevidence that β-glucan can lower serum cholesterol levels, heal wounds,moderate glycaemic response, and alleviate constipation. β-Glucan canactively bind to specific cell receptors and therefore may be useful forthe treatment of a wide variety of disorders or diseases.

[0005] The known methods for extracting β-glucan from cereal grains,such as oats and barley, involve several steps. Firstly, the cerealgrain is milled to a flour prior to extracting β-glucan from the flourusing warm or hot water or an aqueous alkali solution. The milling stepfacilitates release of the β-glucan from the cereal. The aqueous extractof β-glucan is then separated from the solid flour residue. Finally, theβ-glucan is recovered from the extract.

[0006] The known methods of recovering the β-glucan from the aqueousextract include precipitation of the β-glucan using a water misciblesolvent, such as alcohol, or by freezing and then thawing the extract togive a precipitate of β-glucan which can be recovered by filtration orcentrifugation. The extraction of the β-glucan itself from the cereal isnot generally a costly process. However, the recovery of the β-glucanfrom the extract is costly. This is due to the large amounts of waterthat must be removed to give solid β-glucan.

[0007] In addition, it is difficult to control the molecular weight ofthe β-glucan product obtained from known processes. High molecularweight β-glucan is preferable for certain uses. For example, highmolecular weight β-glucan is preferable for moderating glycaemicresponse and for lowering serum cholesterol levels. On the other hand,low molecular weight β-glucan may be preferable as a food additive. Forexample, low molecular weight β-glucan can form a gel having beneficialtextural properties for processed foods.

[0008] In order to obtain a high molecular weight β-glucan product,previous methods of β-glucan extraction from cereals have required thatenzymes present in the cereal be deactivated prior to the extractionstep. The enzymes are responsible for lowering β-glucan molecular weightand are deactivated either by treating the flour with boilingethanol/water mixtures or by treating the flour with an aqueous acidsolution.

SUMMARY OF INVENTION

[0009] It is an object of this invention to provide a process forextracting β-glucan from cereals and to provide a β-glucan productobtained from the process, or at least to provide a useful alternativeprocess or product.

[0010] In one aspect of the invention there is provided a process forobtaining β-glucan from cereal grain including:

[0011] forming flour from the cereal grain;

[0012] mixing the flour with water at a temperature below approximately65° C. to form a slurry of an aqueous solution of β-glucan and a solidresidue;

[0013] separating the aqueous solution from the solid residue;

[0014] removing water from the aqueous solution by evaporation orultrafiltration or combinations thereof to give a concentrated aqueoussolution of β-glucan; and

[0015] forming a β-glucan gel from the concentrated aqueous solution ofβ-glucan.

[0016] Although the cereal used may be any cereal containing β-glucan,the preferred cereal of the invention is barley or oats.

[0017] It is preferred that the gel is formed from the aqueous solutionof β-glucan using any combination of the following steps: shearing,heating, cooling and freezing the solution. Shearing the solution may beby stirring the solution or by passing the solution down a pipe. Thesolution may also be heated and cooled to induce the formation of a gel.It is also preferred that the gel, once formed, is washed with water toremove starch or protein or starch or protein that may have beenhydrolysed. The gel may also be frozen, for example by extrusion into abath containing an aqueous solution of a salt where the temperature ofthe bath is below 0° C. The frozen gel is removed from the bath and thenthawed to give a more compact gel which is more readily isolated byfiltration. The gel may then be dried by, for example, spray drying orhot roller drying.

[0018] Preferably the step of milling the flour is carried out under dryconditions to enable the removal of starch from the cereal. Starchgranules can be removed from the milled flour by sieving or by airclassification. Alternatively, the cereal may be milled in the presenceof either cold water or a mixture of ethanol and water to facilitate theremoval of starch by standard methods.

[0019] The invention therefore also provides a starch rich fractionobtained from the process of this invention and useful as an ingredientin processed foods, for malting, or as a feed for animals.

[0020] It is preferred in the extraction step that the flour is mixedwith water at a temperature greater than 45° C. but less thanapproximately 60° C.

[0021] Preferably the aqueous solution of β-glucan is separated from thesolid residue by centrifugation or by filtration.

[0022] The invention therefore also provides a solid residue obtainedfrom this process and useful as an ingredient in processed foods, formalting, or as a feed for animals.

[0023] The β-glucan is recovered from the aqueous solution by firstlyconcentrating the aqueous solution of β-glucan. Concentration of theaqueous solution may be by evaporation, for example thin-filmevaporation, or ultrafiltration, to form a concentrated aqueoussolution. A β-glucan gel can be formed from this solution. The gel maybe washed with water to remove impurities and then dried, for example byspray drying or hot roller drying, to obtain a β-glucan solid.

[0024] Prior to concentrating the aqueous solution of β-glucan, it ispreferable to remove starch and/or protein impurities. Protein may beremoved by heating the aqueous solution to above about 70° C. causingthe protein to precipitate which can then be removed by filtration or bydecanting or by centrifugation.

[0025] Alternatively, protein may be removed by adding a protease to theaqueous solution followed by ultrafiltration of the degraded protein.Another method of removing protein is to add a flocculant such as acarrageenan, for example κ-carrageenan. Starch may also be removed byadding a starch degrading enzyme, such as an α-amylase, to the aqueoussolution followed by ultrafiltration to remove the degraded starch.

[0026] It is preferable during β-glucan extraction to add an enzyme toreduce the average molecular weight of the β-glucan. The enzyme ispreferably a cellulase (for example, E.C. 3.2.1.4).

[0027] It is also preferable during β-glucan extraction to degrade anyarabinoxylans present by adding an arabinoxylan degrading enzyme, forexample, a xylanase.

[0028] The invention also provides a β-glucan product produced by aprocess of this invention.

[0029] The invention further provides a composition containing β-glucanobtained by a process of this invention.

[0030] The invention also provides a method for lowering serumcholesterol levels in an animal including administering to the animal aβ-glucan product obtained by a process of this invention.

[0031] The invention also provides a method for healing a wound in ananimal including administering to the animal a β-glucan product obtainedby the process of the invention.

[0032] The invention also provides a method for moderating glycaemicresponse in an animal including administering to the animal a β-glucanproduct obtained by the process of this invention.

[0033] The invention also provides a method for alleviating constipationin an animal including administering to the animal a β-glucan productobtained by the process of this invention.

[0034] The invention also provides a method for stimulating the immunesystem in an animal including administering to the animal a β-glucanproduct obtained by the process of this invention.

[0035] The invention also provides a food ingredient containing aβ-glucan product obtained by the process of this invention.

[0036] The invention further provides an edible film as a food coatingprepared using β-glucan obtained by the process of this invention.

DETAILED DESCRIPTION

[0037] β-Glucan occurs naturally in a wide variety of cereals. Theprocess of this invention is not limited to any particular cereal.However, preferred cereals are barley and oats.

[0038] The process of this invention can be varied to give differentβ-glucan products. The physical properties of a β-glucan product aredependent principally on the average molecular weight of the β-glucanmolecules and the conformation of the β-glucan molecules. High molecularweight β-glucan is β-glucan having an average molecular weight greaterthan 5×10⁵ Daltons. Low molecular weight β-glucan is β-glucan having anaverage molecular weight in the range of 5×10³ to 2×10⁵ Daltons.

[0039] β-Glucan products can form a gel in water. The ease with which aβ-glucan product forms a gel depends on the average molecular weight ofthe β-glucan and also depends on the manner in which a solution ofβ-glucan extracted from cereal grain is processed.

[0040] High molecular weight β-glucan is desirable for certaintherapeutic uses because of its high viscosity in aqueous solution. Themoderation of glycaemic response and the lowering of serum cholesterollevels can be effected using β-glucan of high molecular weight. However,enzymes known to degrade β-glucan from high molecular weight β-glucan tolow molecular weight β-glucan are known to be present in cereal grains.Therefore, known methods of obtaining a β-glucan from cereals haverequired an enzyme deactivation step, such as treatment with boilingethanol/water mixtures or by treatment with an aqueous acid solution.

[0041] However, it is known that in some cereals, particularly barley,the β-glucan degrading enzymes are present in the husk and outer layersof the grain. Thus, removal of the husk and outer layers of the grain bypearling leaves a cereal grain which has little or no β-glucan degradingenzyme present. In addition, the outer layers of the grain (the aleuroneand sub-aleurone layers) are depleted in β-glucan. The pearled grain istherefore enriched in β-glucan relative to unpearled grain.

[0042] During aqueous extraction of β-glucan from unpearled grain,colour, flavour, and enzymes from the husks of the grain can appear inthe extract. Following further processing, this can result in a β-glucanproduct having an unacceptable colour or flavour, or being degraded bythe enzymes. Pearling of the grain removes the husks and outer layersand therefore minimises any undesirable colour or flavour of theβ-glucan product.

[0043] During mixing of the flour with water to extract the β-glucan,the water may be at any temperature in the range of 25 to 65° C.However, the temperature of the water is preferred to be approximately45 to 60° C. Preferably the pH of the mixture is in the range 2 to 10.

[0044] Starch is the major constituent of the grain and occurs as smallgranules within the grain. β-Glucan occurs within the cell walls of thegrain which surround the starch granule. The complete or partial removalof starch from flour obtained from the grain would therefore result in afraction enriched in β-glucan. An enriched β-glucan fraction has thefollowing benefits. Firstly, there would be less solid material toremove after the extraction was complete. The extract would contain moreβ-glucan for a given volume of water used. Therefore, less concentrationof the extract would be required. Finally, less starch would besolubilised during the extraction since there is less starch in theflour from which the β-glucan is extracted.

[0045] Various methods are known for complete or partial removal ofstarch from cereal grain. These include dry milling and wet milling. Wetmilling with water has a disadvantage since about 30-50% of the cellwall β-glucan is soluble in water at a temperature of 25° C. However,only 10-20% of the cell wall β-glucan is soluble in ice-cold water.Similarly, little of the cell wall β-glucan is soluble in ethanol orethanol/water mixtures or aqueous solutions of certain salts. Therefore,for wet milling, it is preferable to use cold water or ethanol/watermixtures or aqueous solutions of certain salts.

[0046] Dry milling may be used for removing starch. A large proportionof the starch can be removed from dry flour by sieving or airclassification. The cell wall material containing the β-glucan mostlyoccurs as particles which are larger than the starch granules aftermilling. Consequently, the starch granules will pass through the sievewhile cell wall material will be retained. Air classification willseparate out the dense starch granules from the cell wall material.However, it is to be understood that these methods of separation are not100% efficient and that the starch fraction will contain some cell wallmaterial and the cell wall material will contain some starch.

[0047] The β-glucan from the enriched β-glucan material can now beextracted using hot water. Since there are little or no β-glucandegrading enzymes left in the grain, it can be useful to add an enzyme,preferably a cellulase, to the extraction solution to partially degradethe β-glucan in a controlled fashion. This also helps release theβ-glucan from the enriched material. It can also be advantageous to addan arabinoxylan degrading enzyme, preferably a xylanase, since thesedegrade unwanted arabinoxylans in the extract, decreasing the extractviscosity, increasing the yield of extract after separation from thesolids, and also helping in the release of the β-glucan from theenriched flour.

[0048] After extraction the solids are preferably removed bycentrifugation. The extract can be concentrated at this stage byevaporation of all or some of the water. Such techniques for this arewell known and include thin-film evaporation to obtain a concentratedβ-glucan solution, and spray drying or hot roller drying to obtain aβ-glucan containing solid.

[0049] The final product from this process contains protein and starch.In some cases this less pure form of β-glucan may be the preferredproduct. However, it may be desirable to remove the starch and/orprotein prior to water evaporation to obtain a product of higher purity.Starch can be degraded using a starch degrading enzyme, preferablyα-amylase, and protein can be degraded by a protein degrading enzyme,that is, a protease. The degraded starch and protein can then be removedfrom the extract and the extract concentrated by ultrafiltration. It isalso possible to precipitate the protein by heating the extract aboveabout 70° C. The precipitated protein can then be separated from thesolution of the extract. Heating the extract above 70° C. has theadvantage of also destroying any remaining enzyme activity andsterilising the extract.

[0050] Heating the extract above about 70° C. appears to inhibit gelformation. Heated extracts appear not to form a precipitate when frozenand thawed nor do they gel readily, that is, within a few hours.However, gelation can be induced by the following methods, either aloneor in combination with other methods. Resting the solution for a periodof time, shearing the solution for a period of time, cooling thesolution for a period of time, heating the solution for a period oftime, and freezing the solution for a period of time. Generally it iseasier to induce gelation with more concentrated solutions, especiallythose containing low molecular weight β-glucan.

[0051] Inducing gelation at this stage of the process has severaladvantages over the technique of freezing and then thawing the solution.The expense of freezing in some cases can be avoided. Where freezing isstill required the solutions are more concentrated, thus decreasing thecost of freezing.

[0052] Following gelation it may be preferable to freeze the gel, forexample by extrusion into a bath containing a salt, where the bath is ata temperature of less than 0° C. The gel is then recovered and thawed togive a compact gel which can be more easily filtered.

[0053] Finally after gelation has been induced it can be advantageous towash out the hydrolysed or unhydrolysed starch and protein contaminantsfrom the β-glucan gel before the gel is dried to obtain a β-glucanenriched gel.

[0054] The starch rich fraction obtained from sieving orair-classification could be a valuable product useful in baked andprocessed foods. Similarly, after the extraction of the β-glucan fromthe grain the wet solids remaining contain significant amounts ofβ-glucan. These wet solids could be dried and used in processed foods.The β-glucan in the dried solids could have useful texturisingproperties in a variety of processed foods. It is also possible that thestarch fraction or the wet solids could be used for malting or sold asfeed for animals.

[0055] The invention is described with reference to the followingexamples but is not to be construed as limited thereto.

[0056] In the examples all β-glucan contents were determined using theMegazyme Mixed-Linkage Assay Procedure and the McCleary method or amodification of the McCleary method. The starch andmalto-oligosaccharide contents were determined using the Megazyme TotalStarch Assay Procedure or a modification of this procedure (MegazymeInternational Ireland Ltd, Bray Business Park, Bray, Co. Wicklow,Ireland)

EXAMPLE 1

[0057] Barley grain (50 g) was pearled from 40 to 60% and then finelymilled in a Kenwood mixer with milling attachment. The milled grain wassieved through sieves of sizes 150, 90 and 63 μm. The coarse fractionleft on the sieve was further ground with a mortar pestle and sievedagain. Yields, percentage and absolute β-glucan contents for eachfraction are shown in Table 1. TABLE 1 Fraction Sieve size/μm Yield/gβ-glucan content % β-glucan content/g Coarse >150 12.9 7.61 0.981 Medium150 → 90  6.06 12.53 0.760 Fine 90 → 63 2.32 11 .55 0.268 Very fine  <6325.72 0.85 0.218

[0058] The very fine fraction was the largest fraction sieved butcontained only small amounts of β-glucan. The medium and fine fractionsboth contained about 12% β-glucan. Of the sieved flour fraction 83% ofthe β-glucan occurred in the fraction that sieved between 150 and 60 μm.

EXAMPLE 2

[0059] The release of β-glucan from the medium sieved fraction obtainedin Example 1 was determined in the presence of various enzymes: acellulase (Trichoderma reesei species from Sigma, 6.3 U/ml), xylanase(Shearzyme™ from Novo Nordisk, activity unknown) and protease (Alaclase™from Novo Nordisk, 2.4 AU/g). The medium sieved fraction (1 g, seeExample 1) was added to various combinations of enzymes (see Table 2) inwater (7 ml) and the mixture was heated for 1.5 h at 50° C. to extractβ-glucan. The β-glucan extract was separated from the solids bycentrifuging at 3500 rpm for 10 min and then frozen. After thawing, theyield of precipitated (ppt) β-glucan was determined. The results areshown in Table 2. TABLE 2 Yield of Yield of ppt T. reesei ShearzymeAlaclase extract /ml β-glucan/g 10 ul 10 ul 10 ul 5.67 0.061 5 ul 10 ul0 5.44 0.061 0 10 ul 10 ul 5.73 0.051 5 ul 0 0 5 0.03

[0060] All the enzymes appeared to be effective in increasing the yieldof ppt β-glucan but the Shearzyme™/cellulase combination appeared to bemost effective. The yield of extract after centrifuging was improved byadding Shearzyme™.

EXAMPLE 3

[0061] A flour enriched in β-glucan was prepared by sieving a barleypollard flour. The β-glucan was extracted from the flour by heating amixture of the flour (2 g) with water (10 ml) to which had been addedcellulase (10 μL, Trichoderma reesei species from Sigma, 6.3 U/ml) at50° C. for 30 min. The extract (6.1 ml) was separated from the solids bycentrifuging at 3000 rpm for 15 min. The extract was then heated on aboiling water bath for 5 min to, precipitate protein, which was removedby centrifugation. The extract was evaporated to dryness by rotaryevaporation, which produced a glassy film containing about 53% β-glucan.

EXAMPLE 4

[0062] A barley pollard flour 10 g was mixed with water (50 ml) andheated at 50° C. for 1 h. The extract was separated from the solids bycentrifuging at 3000 rpm for 10 min. This yielded 30 ml of extract. Theextract was then heated to 95° C. for 10 min and the protein thatprecipitated was removed on a centrifuge. The extract was concentratedon rotary evaporator to about 25% of its original volume. The extractwas then stirred rapidly for 2 min to induce shearing and then restedfor 5 min. This procedure was repeated 6 times before the extract wasfrozen for 12 h. No precipitate formed on thawing. Over a period of daysthe solution slowly thickened. After 2 days the solution was frozen andthawed again. This produced a precipitate, which was filtered, washedwith water and dried. The yield was 0.16 g.

EXAMPLE 5

[0063] A pollard flour (30 g) was mixed with water (150 ml) containingShearzyme™ (10 μL, Novo Nordisk, activity unknown,) and cellulase (50μL, Trichoderma reesei species from Sigma, 6.3 U/ml). The mixture washeated at 50° C. for 1.5 h. After 30 min the mixture was found to bereasonably free flowing. A β-glucan extract was recovered from themixture by removing the solids on a centrifuge. The yield of extract was118 ml.

[0064] The extract was treated in a number of ways.

[0065] a) 25 ml of the extract was filtered through glass fibre thentreated with amylase (200 μL, Bacillus species Sigma, 3480U/ml) for 1 h30 min at 30° C. to hydrolyse the starch in the extract. The extract wasthen heated at 90° C. for 15 min and centrifuged (3,000 rpm 10 min) toremove protein and destroy amylase activity. The liquid recovered was 23ml. The extract was dialysed overnight to remove hydrolysed starch. Theextract was then evaporated to an oil in a rotary evaporator and ovendried at 80° C. The β-glucan content of the oven dried material wasabout 57%.

[0066] b) 25 ml of the extract was heated at 90° C. for 15 min thencentrifuged to remove protein. The liquid recovered was 23 ml. Theextract was rotary evaporated to about half its original volume and thendried as a thin film in an oven at 80° C. The β-glucan content of thefilm was about 30%. Approximately 0.2 g of the film was dissolved in 2ml of water at 90° C. to form a transparent solution. The solution wascooled in ice and stirred to induce shearing and then rested. This wasrepeated several times. After leaving overnight a gel had formed. Thegel was frozen. The thawed gel was washed with water and filtered anddried. The gel filtered very readily on a #3 sintered glass filter. Thisyielded 0.066 g of dried gel. The β-glucan content of the dried gel was87%.

[0067] c) 25 ml of the extract was filtered through glass fibre and thentreated with amylase (200 μL, Bacillus species Sigma, 3480U/ml) for 30min at 30° C. to hydrolyse the starch in the extract. The extract washeated at 90° C. for 15 min and centrifuged (3,000 rpm 10 min) to removeprotein and destroy amylase activity. The extract (0.4 ml) was placed inan ultrafiltration centrifugal filter unit (Millipore Ultrafree-MC). Thefilter unit was centrifuged (13,000 rpm for 40 minutes) and about 0.07ml of liquid was recovered which was oven dried to a thin transparentfilm.

EXAMPLE 6

[0068] Barley pollard flour (30 g) was mixed with water (150 ml)containing Shearzyme™ (10 μL, Novo Nordisk, activity unknown) andcellulase (50 μL, Trichoderma reesei species from Sigma, 6.3 U/ml). Themixture was heated on a water bath at 50° C. for 1.5 h. After 30 min themixture was found to be reasonably free flowing. The solids were removedfrom the mixture by centrifuging and the extract that remained washeated at 90° C. for 15 min. The protein that precipitated was removedon a centrifuge. The yield of extract was 118 ml. The extract wasconcentrated to 17 ml by rotary evaporation. A viscous solution remainedwhich was heated to 90° C. and cooled and then heated to 70° C. andcooled. This caused the solution to set rapidly to a soft gel, which wasdispersed in water to remove soluble impurities, and then filtered anddried. The yield of dried gel was 0.71 g. The β-glucan content of thedried gel was 80%. The washings from the gel were rotary evaporated toan oil and then oven dried. This yielded 0.9 g of a glassy material. Theβ-glucan content of the glassy material was 4%. Therefore it appearsthat about 94% of the β-glucan was in the dried gel and only 6% in thegel washings obtained by filtering the gel.

EXAMPLE 7

[0069] The following examples illustrates a novel method for removingstarch which does not result in much solubilisation of β-glucan.

[0070] Removal of starch from the cell-wall material was accomplished byhomogenising in a Kenwood mixer, barley flour (4 g) with water that wassaturated with a salt, in this case sodium sulphate. The solution wasfiltered through a 55 μm nylon mesh. The slurry filtered well,indicating little or no solubilisation of the β-glucan. Remaining on thefilter was the enriched cell-wall fraction (1.75 g) which contained10.4% β-glucan.

EXAMPLE 8

[0071] A gel is formed by concentrating a β-glucan extract. Barley flour(25 g) was mixed with water (175 ml) and a xylanase (6.2 μL, Shearzymefrom Novo Nordisk, activity unknown) and cellulase (125 μL, Penisillumfunicolsum 0.1 mg /ml) was added. The extraction solution was heated at50° C. for 1 h. The extract was separated from the solids bycentrifuging at 3500 rpm for 10 min. The extract was then heated at 90°C. for 10 min to precipitate protein, which was removed by filteringthrough a glass fibre filter. The extract was concentrated to {fraction(1/10)} its original volume and left overnight in the fridge to gel.After heating the gel to 65° C. and then cooling the gel, the gel wasfirmer.

EXAMPLE 9

[0072] More β-glucan can be extracted from finely ground flour thencoarsely ground flour. For each of the medium and the coarse flourfractions prepared in example 1, the flour (0.2 g) was mixed with water(2 ml) to which a xylanase (0.1 μL, Shearzyme from Novo Nordisk,activity unknown) and cellulase (5 μL, Penicillium funicolusum fromSigma, 10 μg/ml) had been added. The extraction was continued at 50° C.for 1 h. The extract was separated from the solids by centrifuging at3500 rpm for 10 min. The β-glucan content of the extract was thenmeasured. For the medium flour fraction about 70% of the β-glucan in theflour was extracted, whereas for the coarse material only about 50% ofthe β-glucan was extracted.

EXAMPLE 10

[0073] Barley flour (5 g ) was mixed with water (35 ml) to which axylanase (Shearzyme from Novo Nordisk, activity unknown) and a cellulase(Celluclast from Novo Nordisk, 1500 NCU/g) had been added according tothe quantities given in Table 3. The extraction solution was heated at50° C. for 2 h. The extract was separated from the solids bycentrifuging at 3500 rpm for 10 min. The extract was then heated at 90°C. for 10 min to precipitate protein, which was removed by centrifuging.After a freeze/thaw of the extract the precipitate of β-glucan solids inthe thawed liquid was filtered, and washed with water then ethanol. Thesolids were dried and the viscosity of a 1% solution measured. Mw, theweight average molecular weight was estimated from the viscosity usingthe method of Böhm, N. and Kulicke, W-M. Carbohydr. Res. 315 (1999)293-301, and are shown in Table 3. TABLE 3 Shearzyme Celluclast Relativeadded/μL added/μL viscosity Mw 2 0.2  49 19000 2 0.02 123 75000 2 0 300194000 

[0074] The molecular weight thus can be altered by changing thequantities of β-glucan degrading enzyme added to the reaction mixture.

EXAMPLE 11

[0075] It is advantageous to use cold-water to wash out the starch andcause minimum solubilisation of the β-glucan. Barley flour (0.2 g)containing 8.5% β-glucan was mixed with water (2 ml) at 4.5° C. for 2 h.The extract was separated from the solids by centrifuging at 3500 rpmfor 10 min. From the β-glucan content of the extract it was calculatedthat only about 5% of the β-glucan in the flour was solubilised.

EXAMPLE 12

[0076] For maximum protein precipitation the pH of the extract should benear the isoelectric point of the protein. Barley flour (10 g) was mixedwith water (70 ml) and the extract mixture was heated at 50° C. for 1 h.The extract was separated from the solids by centrifuging at 3500 rpmfor 10 min. A portion (5 ml) of the extract was taken and the pHadjusted to 7.0 with NaOH solution (0.1 M). On heating to 95° C. noprotein precipitation was observed.

EXAMPLE 13

[0077] To decrease the amounts of starch and maltodextrins in theextracts it is advantageous to deactivate partially or completely thenative amylases in the flour, which improves the purity and gelproperties of the β-glucan. Acid treatment and heating was found to beeffective in deactivating the amylases.

[0078] A solution of the amylase was prepared by mixing barley flour (20g) with water (200 ml) and immediately centrifuging the mixture. Thesupernatant was then filtered with Glass fibre (Watman GF/A) to removefines. The supernatant was then treated by adjusting the pH and heating.Amylase activity of the supernatant was measured by mixing an equalamount of the treated supernatant with a potato starch solution (1.5%)and recording the decrease in viscosity. Results are shown in Table 4.TABLE 4 Treatment Viscosity Viscosity Viscosity Viscosity Code ofsupernatant after 1 min after 5 min after 10 min after 20 min A None 7063 59 55 B Heated at 95° C. 87 90 89 88 for 15 min C PH adjusted to 3.890 90 89 86 then heated at 50 ° C. for 25 min, pH then adjusted to 5.4.

EXAMPLE 14

[0079] Protein precipitation by addition of a precipitating agent suchas carrageenan can be useful for removing additional amounts of protein.This improves the purity and gelling properties of the β-glucan. Foroptimum protein precipitation the pH of the solution should be below theisoelectric point of the protein.

[0080] A flour from a pearled barley (5 g) was mixed with water (35 ml)to which had been added a xylanase (2 μL, Shearzyme from Novo Nordisk)and a cellulase (0.05 μL. Celluclast from Novo Nordisk, 1500 NCU/g).Dilute HCl (200 μL, 0.1 M) and carrageenan (150 μL, 1%, Viscarin BF 136Cfrom FMC) was added. A brown precipitate forms which was removed bycentrifuging.

EXAMPLE 15

[0081] Sieved barley fractions were prepared enriched in β-glucan.Barley (5.1% β-glucan content) was pearled to a weight loses of 30%. Thegrain was milled on the finest setting of a Kenwood mixer fitted with agrain mill attachment. Of the flour formed, 5 g was hand sifted throughtwo successive sieves containing a 150 and 63 micron mesh. The coarsematerial retained as the over on the 150 micron sieve was ground in amortar and pestle until most past through the 150 micron sieve. Threefractions were obtained as shown in Table 5. TABLE 5 Sieve β-glucanβ-glucan % Code size/micron Yield content/% of total a >150 0.20 9.4 9 b150 → 63 0.83 16.8 65 c  <63 3.33 1.7 26

EXAMPLE 16

[0082] The stability of a gel that had been frozen was tested byrepeated washings with water. The gel (4.9 g) was filtered on a 55 μmmesh to remove excess water and the filtrate retained. The gel was thenwashed with water (10 ml) and the second filtrate retained. The β-glucancontent of the filtrates and gel were measured. Results shown in thetable indicate little solubilisation of β-glucan in the gel duringwashing. TABLE 6 Sample β-glucan content/% % of total β-glucan Firstfiltrate 0.15  2% Second filtrate 0.014 0.7% Gel 11.7 97%

EXAMPLE 17

[0083] Amylase deactivation lessens the amount of maltose and starchsolubilised during extraction. Water (10 ml) was adjusted to pH=2.4 withHCl (˜0.65 ml, 1.0 M) and added to flour (10 g) milled from a pearledbarley. For this mixture the pH was found to be 2.8. The mixture washeated at 50° C. for 20 min on a water bath to deactivate the amylase.The pH of the mixture was then adjusted to 5.5 with NaOH (2 ml, 1.0 M).A xylanase (4 μL, Shearzyme Novo Nordisk) and a cellulase (0.1 μLCelluclast from Novo Nordisk) was added to the extraction mixture andthe extraction continued for 1 h. The mixture was then centrifuged at(3000 rpm, 5 min) and the supernatant retained. The solution was thenacidified with HCl (1.6 ml, 0.1 M) and κ-carrageenan (1.2 ml, 1%) wasadded. The precipitate that was formed was removed by centrifuging togive a bright solution. The solution was lyophilised to a whitish solid.The above experiment was repeated, but no enzyme deactivation step wasincluded.

[0084] The starch/malto-oligosaccharide content of the solids with andwithout amylase deactivation was 9% and 26%, respectively.

EXAMPLE 18

[0085] An extract was formed from flour obtained from pearled barleyaccording to Example 17 (with amylase deactivation). The solutionobtained after protein precipitation was rotary evaporated to an oil andleft at 4° C. for 2 days. During this time the oil set to a gel, whichwas washed several times with water. The gel was pressed between papertowels to remove excess water. Solids content of the gel was 17% ofwhich 75.6% was β-glucan. Some 50% of the β-glucan in the flour wasrecovered in the gel.

EXAMPLE 19

[0086] Barley (1000 kg) was pearled to produce pearl barley (700 kg).The pearl barley was milled through two roller mills and a hammer milland then screened to produce two flour fractions. The first fraction(420 kg) contained approximately 80% of the β-glucan. The secondfraction (280 kg) contained approximately 20% of the β-glucan. Thesecond fraction was discarded. The first fraction was divided into sevenbatches (each 69 kg).

[0087] Each batch was mixed into warm water (1200 L) to give a mixtureat a temperature of 50° C. Cellulase (0.5 ml, Celluclast from NovoNordisk, 1500 NCU/g) and Xylanase (60 ml, Shearzyme from Novo Nordisk,activity unknown) enzymes were added to the mixture which was stirredand held for 60 minutes. The mixture was then passed through a solidbowl decanter and a centrifugal clarifier to remove all insolublematerial. The insoluble material was discarded.

[0088] The resulting liquid extract (900 L) was adjusted to a pH of 4.5.Amyloglucosidase enzyme (150 ml, AMG 300 L from Novo Nordisk, 300 ACU/g)was then added to hydrolyse any soluble starch. After the extract wasshown to be starch negative, it was heated to 95° C. for 15 minutes andthen centrifuged to remove the insoluble protein.

[0089] The extract from all seven batches was combined and filteredthrough a diatomaceous earth filter. The filtered extract was thenconcentrated in a triple effect falling film evaporator, followed by asingle effect scraped surface evaporator, to approximately 12% totalsolids. The concentrate was then cooled at less than 0° C. for 24 hoursto develop a suitable gel structure. The gel was then washed in coldwater to remove the remaining soluble sugars and other soluble material.

[0090] The gel was recovered from the mixture using a centrifugalclarifier and then dried in a spray drier to approximately 5% moistureto give β-glucan powder (14 kg). The powder was a fine, free flowingpale cream β-glucan powder. The β-glucan content was approximately 85%on a dry solids basis and had a molecular weight of approximately50,000d.

EXAMPLE 20

[0091] A liquid extract was prepared according to Example 19 above butwas subjected to ultrafiltration following filtration through thediatomaceous earth filter, rather than concentration in a triple effectfalling film evaporator.

[0092] The extract (500 L) was collected after diatomaceous earthfiltration and was purified and concentrated using an ultrafiltrationmembrane. The extract was circulated through a spiral typeultrafiltration membrane. The membrane had an area of approximately 6.4sq. metres and a nominal molecular cut-off of 10kd. Circulation wascontinued until the volume of the circulate was reduced to 100 L. Water(100 L) was added and the circulation was continued until the volume wasreduced again to 100 L. At the end of the process, 80% of the liquid hadbeen removed as permeate and the β-glucan purity had increased from 35%to 60% of total solids.

[0093] Although the invention has been described by way of example, itshould be appreciated that variations and modifications may be madethereto without departing from the invention.

[0094] Furthermore, where known equivalents exist to specific features,such equivalents are incorporated as if specifically set forth herein.

[0095] Industrial Applicability

[0096] The β-glucan products of this invention are useful as foodadditives and as therapeutic agents. They provide desirable texture tofoods, can be used as edible films for food coatings, and can be used asbulking agents in foods. The products of the invention are also usefulas therapeutic agents including agents for lowering serum cholesterollevels, healing wounds, moderating glycaemic response, alleviatingconstipation, and stimulating the immune system.

1. A process for obtaining β-glucan from cereal grain including: formingflour from the cereal grain; mixing the flour with water at atemperature below approximately 65° C. to form a slurry of an aqueoussolution of β-glucan and a solid residue; separating the aqueoussolution from the solid residue; removing water from the aqueoussolution by evaporation or ultrafiltration or combinations thereof togive a concentrated aqueous solution of β-glucan; and forming a β-glucangel from the concentrated aqueous solution of β-glucan.
 2. A process asclaimed in claim 1 wherein the flour is formed by pearling the cerealgrain to remove the husk and outer layers of the cereal grain and thenmilling the pearled cereal grain.
 3. A process as claimed in claim 1 orclaim 2 wherein the pH of the water is adjusted either before or aftermixing with the flour.
 4. A process as claimed in claim 3 wherein the pHis adjusted to less than approximately 4.0 and the slurry is then heatedto greater than approximately 40° C. for at least approximately 10minutes to deactivate amylases in the flour.
 5. A process as claimed inclaim 5 wherein the pH is readjusted to greater than approximately 4.0after heating.
 6. A process as claimed in any one of the precedingclaims wherein the flour is mixed with water at a temperature of 45° C.to 60° C.
 7. A process as claimed in any one of the preceding claimswherein the flour is mixed with water at a temperature of approximately50° C.
 8. A process as claimed in any one of the preceding claimswherein the flour is mixed with water at a temperature of approximately50° C. for 15 to 60 minutes.
 9. A process as claimed in any one of thepreceding claims further including adding an enzyme to the slurry todegarde any arabinoxylans which may be present in the slurry.
 10. Aprocess as claimed in claim 9 wherein the enzyme is a xylanase.
 11. Aprocess as claimed in any one of the preceding claims where an enzyme isadded to the slurry to assist the release of β-glucan from the flour.12. A process as claimed in claim 11 wherein the enzyme is a cellulase.13. A process as claimed in any one of the preceding claims wherein theaqueous solution is separated from the solid residue by centrifugationfollowed by decantation or by filtration.
 14. A process as claimed inany one of the preceding claims wherein an enzyme is added to theaqueous solution to degrade starch.
 15. A process as claimed in claim 14where the enzyme is an α-amylase.
 16. A process as claimed in any one ofthe preceding claims wherein an enzyme is added to the slurry or to theaqueous solution to reduce the average molecular weight of the β-glucan.17. A process as claimed in claim 16 wherein the enzyme is a cellulase.18. A process as claimed in any one of the preceding claims wherein aprotease is added to the aqueous solution to degrade proteins.
 19. Aprocess as claimed in any one of the preceding claims further includingheating the aqueous solution to precipitate protein.
 20. A process asclaimed in any one of the preceding claims further including adding aflocculant to the aqueous solution to precipitate protein.
 21. A processas claimed in claim 20 wherein the flocculant is carrageenan.
 22. Aprocess as claimed in any one of the preceding claims further includingdrying the gel.
 23. A process as claimed in claim 22 wherein the gel isdried by spray drying or by hot roller drying.
 24. A process as claimedin any one of the preceding claims further including inducing shearingin the aqueous solution to assist formation of the gel.
 25. A process asclaimed in any one of the preceding claims further including heating andthen cooling the aqueous solution to assist formation of the gel.
 26. Aprocess as claimed in any one of the preceding claims further includingfreezing the gel and then thawing to increase the density or compactnessof the gel.
 27. A process as claimed in any one of the preceding claimsfurther including washing the gel with water to remove impurities, suchas starch or protein or fragments thereof.
 28. A process as claimed inany one of the preceding claims further including removing impuritiesfrom the aqueous solution by ultrafiltration and diafiltration.
 29. Aprocess as claimed in any one of the preceding claims wherein the flouris formed by milling the cereal grain under dry conditions and thenremoving starch granules by sieving or by air classification.
 30. Aprocess as claimed in any one of the preceding claims wherein the flouris formed by milling the cereal grain in the presence of either coldwater or a mixture of ethanol and water, or an aqueous salt solution,and then removing starch.
 31. A process as claimed in any one of thepreceding claims wherein the cereal is barley or oats.
 32. A β-glucangel obtained by the process of any one of the preceding claims.
 33. Aβ-glucan solid obtained by the process of claim
 22. 34. A compositioncontaining a β-glucan gel obtained by the process of any one of claims 1to
 31. 35. A composition containing a β-glucan solid obtained by theprocess of claim
 22. 36. The use of a β-glucan gel obtained by theprocess of any one of claims 1 to 31 as a food ingredient.
 37. The useof a β-glucan solid obtained by the process of claim 22 as a foodingredient.
 38. A food which contains a β-glucan obtained as a gel bythe process of any one of claims 1 to
 31. 39. A food which contains aβ-glucan obtained as a solid by the process of claim
 22. 40. A starchrich fraction obtained by the process of claim 29 or claim
 30. 41. Afood containing the starch rich fraction of claim
 40. 42. A method forlowering serum cholesterol levels in an animal including administeringto the animal β-glucan obtained by the process of any one of claims 1 to31.
 43. A method for healing a wound in an animal includingadministering to the animal β-glucan obtained by the process of any oneof claims 1 to
 31. 44. A method for moderating glycaemic response in ananimal including administering to the animal β-glucan obtained by theprocess of any one of claims 1 to
 31. 45. A method for alleviatingconstipation in an animal including administering to the animal β-glucanobtained by the process of any one of claims 1 to
 31. 46. A method forstimulating the immune system in an animal including administering tothe animal β-glucan obtained by the process of any one of claims 1 to31.